Opportunities for improving biodiesel production via lipase catalysis

Filamentous fungi are harnessed as cell factories for the production of a diverse range of organic acids, proteins, and secondary metabolites. Growth and morphology have critical implications for product titres in both submerged and solid-state fermentations. Recent advances in systems-level understanding of the filamentous lifestyle and development of sophisticated synthetic biological tools for controlled manipulation of fungal genomes now allow rational strain development programs based on data-driven decision making. In this review, we focus on Aspergillus spp. and other industrially utilised fungi to summarise recent insights into the multifaceted and dynamic relationship between filamentous growth and product titres from genetic, metabolic, modelling, subcellular, macromorphological and process engineering perspectives. Current progress and knowledge gaps with regard to mechanistic understanding of product secretion and export from the fungal cell are discussed. We highlight possible strategies for unlocking lead genes for rational strain optimizations based on omics data, and discuss how targeted genetic manipulation of these candidates can be used to optimise fungal morphology for improved performance. Additionally, fungal signalling cascades are introduced as critical processes that can be genetically targeted to control growth and morphology during biotechnological applications. Finally, we review progress in the field of synthetic biology towards chassis cells and minimal genomes, which will eventually enable highly programmable filamentous growth and diversified production capabilities. Ultimately, these advances will not only expand the fungal biotechnology portfolio but will also significantly contribute to a sustainable bio-economy.

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Moulding the mould: understanding and reprogramming filamentous fungal growth and morphogenesis for next generation cell factories

Citric acid is the world’s largest consumed organic acid and is widely used in beverage, food and pharmaceutical industries. Aspergillus niger is the main industrial workhorse for citric acid production. Since the release of the genome sequence, extensive multi-omic data are being rapidly obtained, which greatly boost our understanding of the citric acid accumulation mechanism in A. niger to a molecular and system level. Most recently, the rapid development of CRISPR/Cas9 system facilitates highly efficient genome-scale genetic perturbation in A. niger. In this review, we summarize the impact of systems biology on the citric acid molecular regulatory mechanisms, the advances in metabolic engineering strategies for enhancing citric acid production and discuss the development and application of CRISPR/Cas9 systems for genome editing in A. niger. We believe that future systems metabolic engineering efforts will redesign and engineer A. niger as a highly optimized cell factory for industrial citric acid production.

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Systems metabolic engineering for citric acid production by Aspergillus niger in the post-genomic era.

Pichia pastoris is extensively used to produce various heterologous proteins. Amounts of biopharmaceutical drugs and industrial enzymes have been successfully produced by fed-batch high-cell-density fermentation (HCDF) of this cell factory. High levels of cell mass in defined media can be easily achieved and therefore large quantities of recombinant proteins with enhanced activities and lower costs can be obtained through HCDF technology. A robust HCDF process makes a successful transition to commercial production. Recently, efforts have been made to increase the heterologous protein production and activity by the HCDF of P. pastoris. However, challenges around selecting a suitable HCDF strategy exist. The high-level expression of a specific protein in P. pastoris is still, at least in part, limited by optimizing the methanol feeding strategy. Here, we review the progress in developments and applications of P. pastoris HCDF strategies for enhanced expression of recombinant proteins. We focus on the methanol induction strategies for efficient fed-batch HCDF in bioreactors, mainly focusing on various stat-induction strategies, co-feeding, and the limited induction strategy. These processes control strategies have opened the door for expressing foreign proteins in P. pastoris and are expected to enhance the production of recombinant proteins.

Fed-batch high-cell-density fermentation strategies for Pichia pastoris growth and production.

Microbial citric acid has high economic importance and widely used in beverage, food, detergents, cosmetics and pharmaceutical industries. The filamentous fungus Aspergillus niger is a work horse and important cell factory in industry for the production of citric acid. Although in-depth literatures and reviews have been published to explain the biochemistry, biotechnology and genetic engineering study of citric acid production by Aspergillus niger separately but the present review compiled, all the aspects with upto date brief summary of the subject describing microorganisms, substrates and their pre-treatment, screening, fermentation techniques, metabolic engineering, biochemistry, product recovery and numerous biotechnological application of citric acid for simple understanding of microbial citric acid production. The availability of genome sequence of this organism has facilitated numerous studies in gene function, gene regulation, primary and secondary metabolism. An attempt has been also made to address the molecular mechanisms and application of recent advanced techniques such as CRISPR/Cas9 systems in enhancement of citric acid production.

Citric acid from Aspergillus niger: a comprehensive overview

The mycelial morphology of Aspergillus niger, a major filamentous fungus used for citric acid production, is important for citric acid synthesis during submerged fermentation. To investigate the involvement of the chitin synthase gene, chsC, in morphogenesis and citric acid production in A. niger, an RNAi system was constructed to silence chsC and the morphological mutants were screened after transformation. The compactness of the mycelial pellets was obviously reduced in the morphological mutants, with lower proportion of dispersed mycelia. These morphological changes have caused a decrease in viscosity and subsequent improvement in oxygen and mass transfer efficiency, which may be conducive for citric acid accumulation. All the transformants exhibited improvements in citric acid production; in particular, chsC-3 showed 42.6% higher production than the original strain in the shake flask. Moreover, the high-yield strain chsC-3 exhibited excellent citric acid production potential in the scale-up process.The citric acid yield and the conversion rate of glucose of chsC-3 were both improved by 3.6%, when compared with that of the original strain in the stirred tank bioreactor.

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing.

When phospholipase A 1 (PLA 1 ) was bio-imprinted with 5% soybean lecithin at pH 5.0, its phospholipase (PLA) activity in lecithin–hexane solution increased by 30.9-fold and its substrate specificity was substantially enhanced. A further increase in PLA activity of the bio-imprinted PLA 1 (bi-PLA 1 ) from 562 U/g to 1288 U/g was obtained when 105 mg diatomite/mg bi-PLA 1 was incorporated as an immobilization carrier. The degumming abilities of PLA 1 , bi-PLA 1 , and immobilized bi-PLA 1 (im-bi-PLA 1 ) in crude soybean oil and crude oil–hexane (1:1 w/w) miscella were also investigated. The phosphorus content in the crude soybean oil was reduced to 7.3 mg/kg from an initial level of 406.9 mg/kg after incubation with 100 mg/kg im-bi-PLA 1 and 2% water at 50 °C for 180 min. In miscella degumming, the residual phosphorus content was reduced from 406.9 mg/kg to 18.7 mg/kg by incubation with 100 mg/kg im-bi-PLA 1 and 2% water at 50 °C for 180 min and neutralization with 3 M NaOH solution. In comparison, the original PLA 1 reduced the residual phosphorus content to 62.9 mg/kg and 92.4 mg/kg in crude oil and miscella degumming, respectively.

Enhancing the performance of a phospholipase A1 for oil degumming by bio-imprinting and immobilization

Prenylated quinones, especially menaquinones, have significant physiological activities, but are arduous to synthesize efficiently. Due to the relaxed aromatic substrate specificity and prenylation regiospecificity at the ortho- site of the phenolic hydroxyl group, the aromatic prenyltransferase NovQ from Streptomyces may be useful in menaquinone synthesis from menadione. In this study, NovQ was overexpressed in Pichia pastoris. After fermentation optimization, NovQ production increased by 1617%. Then the different effects of metal ions, detergents and pH on the activity of purified NovQ were investigated to optimize the prenylation reaction. Finally, purified NovQ and cells containing NovQ were used for menadione prenylation in vitro and in vivo, respectively. Menaquinone-1 (MK-1) was detected as the only product in vitro with γ,γ-dimethylallyl pyrophosphate and menadione hydroquinol substrates. MK-3 at a concentration of 90.53 mg/L was detected as the major product of whole cell catalysis with 3-methyl-2-buten-1-ol and menadione hydroquinol substrates. This study realized whole cell catalysis converting menadione to menaquinones.

Biotransformation of menadione to its prenylated derivative MK-3 using recombinant Pichia pastoris.

By comparing the survival rate and positive mutation rate of the primary mutagenic strain and progeny mutagenic strain under different radiation doses, the results showed that the tolerance of the mutagenic strain to radiation dose increased with the increase of the mutagenic generations. We adopted an improved gradient radiation breeding strategy to improve the breeding efficiency. The strains were treated with radiation in four stages. The first stage was low energy N+ ion implantation (ion energy 15 keV, dose 80 × 2.6 × 1013 cm−2). In the second stage, the energy and dose of N+ ion reached to 20 keV, 90 × 2.6 × 1013 cm−2. In the third stage, 60Co-γ radiation (dose of 1.56 kGy) was used. In the fourth stage, the radiation dose of 60Co-γ increased to 1.82 kGy. After each stage of radiation, the MK (Menaquinone) precursor 1, 4-dihydroxy-2-naphthalate (DHNA) was used as the stress factor to domesticate the mutant strains. By gradually increasing the concentration of DHNA in the culture medium, the substrate tolerance of Flavobacterium sp. was effectively improved. By measuring SOD (superoxide dismutase) activity and malondialdehyde, it showed that the cell damage caused by radiation mutagenesis to the offspring mutant was less than that of the primary mutant. Changes in membrane permeability and membrane potential of the mutant strains were reflected in changes in fluorescence intensity of luciferin diacetate and rhodamine 123, which could explain the enhanced substrate tolerance of strain F-2. After gradient radiation breeding and culture acclimation, the biomass of mutant Strain F-2 was 6.59 g/L, and the MK yield was 9.59 mg/L.

Xiaowen Sun

Papers

Morphological regulation of Aspergillus niger to improve citric acid production by chsC gene silencing.

Enhancing the performance of a phospholipase A1 for oil degumming by bio-imprinting and immobilization

Biotransformation of menadione to its prenylated derivative MK-3 using recombinant Pichia pastoris.

Gradient radiation breeding and culture domestication of menaquinone producing strains