Synthesis of carbon-14 and stable isotope labeled Censavudine.

Censavudine is a nucleoside reverse transcriptase inhibitor (NRTI) explored clinically by Bristol Myers Squibb for the treatment of human immunodeficiency virus-1 (HIV-1). As part of the development process, a carbon-14 labeled analog was synthesized for use in a human absorption, distribution, metabolism, and excretion (ADME) study. A stable isotope labeled analog was also synthesized for use as a mass spectrum internal standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Censavudine was synthesized in 10 steps in a 9% overall yield from carbon-14 labeled trimethylsilylacetylene. A total of 4.44 mCi of material was prepared with a specific activity of 0.25 µCi/mg. The radiochemical and UV purities were 99% and it met all of the specifications for use in a human clinical study. Deuterium labeled Censavudine was synthesized in two steps in a 68% overall yield from [D4 ]-thymine. A total of 237 mg were prepared with a UV purity of 99%.

Anaerobically Digesting Hazardous Waste Pichia pastoris Associated with Butyric Acid Cleaner Production.

Recombinant Pichia pastoris semisolid hazardous waste treatment is difficult and traditional solid waste treatment is not applicable. However, P. pastoris wastes have features of high density and enriched proteins/polysaccharides, which could supply nitrogen/carbon sources for butyric acid production. The waste P. pastoris was first treated using NaOH to form a waste yeast suspension, and then the suspension was mixed with glucose to obtain a starting medium containing 5.6 g DCW/L (dry cell weight) yeast to initiate butyrate fermentation. The suspension was intermediately supplemented to bring the total waste yeast concentration to 26.3 g DCW/L while continuously feeding the concentrated glucose solution. With the proposed strategy, butyrate concentration reached high levels of 51.0-54.0 g/L using Clostridium tyrobutyricum as the strain. Amino acids/oligosaccharides/SO4 2- in the suspension, raw material costs, complicated pretreatment process, and butyric acid cleaner production could be effectively utilized, reduced, eliminated, and realized. However, the apparent waste P. pastoris reduction rate was only 49% per batch, thus a "tanks in-series type's repeated waste treating system" model was developed to theoretically explore the possibility of increasing the waste yeast reduction rate R. The simulation results indicated that when setting the treatment unit numbers at 4, waste solid concentration could decrease from 26.3 to 3.37 g DCW/L and the hazardous waste yeast reduction rate R would increase from 49 to 97%.

High butanol/acetone ratio featured ABE production using mixture of glucose and waste Pichia pastoris medium-based butyrate fermentation supernatant.

In this study, butanol (ABE) fermentations were implemented in a 7 L anaerobic fermentor, by directly using the mixture of glucose solution with the corn/waste Pichia pastoris medium-based butyrate fermentation supernatants (BFS II) as the co-substrate, followed by consecutively feeding of the BFS and concentrated glucose solution. When compared with the major index of ABE fermentation using 150 g/L corn-based medium, butanol concentration could be maintained at high level of 12.7-12.8 g/L, butanol/acetone (B/A) largely increased from ~ 2.0 to 4.4-5.0, butanol yield on total carbon sources increased from 0.32-0.34 to 0.39-0.41 (mol base) with a higher butyrate/glucose consumption ratio of 37%-53%. Efficient utilization of butyrate, SO42-, amino acids, oligosaccharides, etc. in BFS II and the intracellular NADH contributed to the ABE fermentation performance improvement. The proposed strategy could be considered as the second utilization of waste Pichia pastoris, which could save raw materials/operating costs, fully use the oligosaccharides/SO42- in BFS II to relieve the working loads in downstream waste water treatment process, and increase fermentation products diversity/flexibility to deal with the varied marketing prices and requirements.

MiR-379 relieves myocardial injury after acute myocardial infarction by regulating tumor necrosis factor-α-induced protein 8.

BACKGROUND: Acute myocardial infarction (AMI) is the myocardial avascular necrosis syndrome caused by coronary atherosclerotic plaque rupture, thrombosis or coronary artery occlusion. Therefore, it is of great significance to find new targets for the treatment of myocardial infarction. The purpose of this study was to investigate the effect of microRNA-379 (miR-379) on AMI and its mechanism. METHODS: MiR-379 mimic was used to transfect H9c2 cells and we determined the protective effect of miR-379 on H9c2 by detecting the level of apoptosis. TargetScan software was used to detect miR-379's downstream targets. We constructed siRNA to analyze the effect of miR-379's downstream targets on H9c2 cells. In addition, we used miR-379 agomir to inject the tail vein of AMI rats to verify the effect of miR-379 on rat cardiomyocytes. RESULTS: TargetScan detected that miR-379 and Tumor necrosis factor-α-induced protein 8 (TNFAIP8) may have binding sites and the dual luciferase reporter assay found that miR-379 binds to TNFAIP8 and inhibits its activity. MiR-379 mimic was found to reduce the expression of caspase3 and caspase9 in H9c2 cells and thereby reduce H2O2-induced cell damage. Inhibition of TNFAIP8 also significantly reduced apoptosis level and inhibited the NF-κB signaling pathway in H9c2 cells. Finally, miR-379 agomir was used to inject the tail vein of AMI rats and verified the protective effect of miR-379 in the heart in vivo. CONCLUSIONS: MiR-379 has a binding site with TNFAIP8 and can inhibit its activity by binding to TNFAIP8 mRNA. SiRNA-TNFAIP8 can inhibit the NF-κB signaling pathway and protect myocardial cells from AMI-induced myocardial damage by reducing the apoptosis level of myocardial cells.

Predictive value of miRNA-126 on in-stent restenosis in patients with coronary heart disease: A protocol for meta-analysis and bioinformatics analysis.

BACKGROUND: In-stent restenosis (ISR) is one of the most important complications and impacts the long-term effects after percutaneous coronary intervention (PCI) in patients with coronary heart disease (CHD). Related studies have revealed that microRNA (miRNA) can predict ISR in CHD patients. MiRNA-126 may be a potential biomarker for the diagnosis of ISR. However, the accuracy of miRNA-126 in the diagnosis of ISR is still controversial. Therefore, this study carried out meta-analysis to further evaluate the accuracy of miRNA-126 in the diagnosis of ISR. At the same time, bioinformatics is used to predict the target genes and miRNA-126 may be involved in regulation, so as to provide theoretical support for the precise treatment of CHD. METHODS: The literatures on the miRNA-126 diagnosis of ISR in CHD patients were collected by searching on computer through China National Knowledge Infrastructure, Wanfang, China Biology Medicine disc, PubMed, EMBASE, Cochrane Library and Web of Science. The retrieval time is set to build the database until April 2021. The meta-analysis of the literatures that meet the quality standards was conducted by Stata 16.0 software. TargetScan database, PicTar database, miRanda database, and miRDB database were used to predict miRNA-126 intersection target genes. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment analysis of miRNA-126 target genes were performed by using DAVID database. STRING database was applied to analyze the protein-protein interaction (PPI) network of miRNA-126 target genes. The "Networkanalyzer" function of Cytoscape3.7.2 software is adopted to analyze the network topology attributes, so as to find out the core genes of PPI network. RESULTS: The results of this meta-analysis will be submitted to a peer-reviewed journal for publication. CONCLUSION: In this study, meta-analysis and bioinformatics analysis were adopted to further evaluate the accuracy of miRNA-126 in the diagnosis of ISR in CHD patients, and to explore the mechanism of the action of miRNA-126 and understand related pathways. ETHICS AND DISSEMINATION: The private information from individuals will not be published. This systematic review also should not damage participants' rights. Ethical approval is not available. The results may be published in a peer-reviewed journal or disseminated in relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/9FMR5.

Predictive value of miRNA-21 on coronary restenosis after percutaneous coronary intervention in patients with coronary heart disease: A protocol for systematic review and meta-analysis.

BACKGROUND: Evidence reveals that microRNA (miRNA) can predict coronary restenosis in patients suffering from coronary heart disease (CHD) after percutaneous coronary intervention (PCI). Perhaps, miRNA-21 is a promising biomarker for the diagnosis of coronary restenosis after PCI. However, the accuracy of miRNA-21 has not been systematically evaluated. Therefore, it is necessary to perform meta-analysis to certify the diagnostic values of miRNA-21 on coronary restenosis after PCI. METHODS: China National Knowledge Infrastructure, Wanfang, VIP, and China Biology Medicine disc, PubMed, EMBASE, Cochrane Library, and Web of Science were searched for relevant studies to explore the potential diagnostic values of miRNA-21 on coronary restenosis after PCI from inception to January 2021. All data were extracted by 2 experienced researchers independently. The risk of bias about the meta-analysis was confirmed by the Quality Assessment of Diagnostic Accuracy Studies-2. The data extracted were synthesized and heterogeneity was investigated as well. All of the above statistical analyses were carried out with Stata 16.0. RESULTS: This study proved the pooled diagnostic performance of miRNA-21 on coronary restenosis after PCI. CONCLUSION: This study clarified confusions about the specificity and sensitivity of miRNA-21 on coronary restenosis after PCI, thus further guiding their promotion and application. ETHICS AND DISSEMINATION: Ethical approval is not required for this study. The systematic review will be published in a peer-reviewed journal, presented at conferences, and shared on social media platforms. This review would be disseminated in a peer-reviewed journal or conference presentations. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/356QK.

Relationship between A1166C polymorphism of angiotensin II type 1 receptor gene and arteriosclerosis: A protocol for systematic review and meta-analysis.

BACKGROUND: Arteriosclerosis has genetic correlation. Many studies have shown that angiotensin II type 1 receptor (AT1R) gene A1166C polymorphism is highly associated with arteriosclerosis, but there is no evidence-based basis. The purpose of this study is to systematically evaluate the relationship between AT1R gene A1166C polymorphism and arteriosclerosis. METHODS: The search time is set from the establishment of the database in December 2020 in this study. The search database include China National Knowledge Infrastructure (CNKI), Wanfang, VIP and China Biology Medicine disc (CBM), PubMed, EMBASE, Web of Science, and the Cochrane Library. The subjects are observational studies on the relationship between AGTR1 A1166C polymorphism and arteriosclerosis (including case-control study, cross-sectional study, and cohort study). The language is limited to English and Chinese. The data of the included study are extracted and the literature quality is evaluated by 2 researchers independently. The data are statistically analyzed by Stata 16.0 software. RESULTS: This study will use pulse wave velocity as an index to evaluate arteriosclerosis to explore the relationship between AT1R gene A1166C polymorphism and arteriosclerosis. CONCLUSION: This study will provide evidence-based medicine for elucidating the genetic tendency of arteriosclerosis. ETHICS AND DISSEMINATION: Private information from individuals will not be published. This systematic review also does not involve endangering participant rights. Ethical approval will not be required. The results may be published in a peer-reviewed journal or disseminated at relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/V6E2Y.

[Expression optimization and molecular modification of heparin C5 epimerase].

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.

Matrix metalloproteinase-13, NF-κB p65 and interleukin-1ß are associated with the severity of knee osteoarthritis.

Knee osteoarthritis (KOA) is a prevalent disease, especially in the elderly. The present study examined the expression of matrix metalloproteinase-13 (MMP-13), NF-κBp65 and interleukin (IL)-lß in the synovial tissues of KOA patients and the role of MMP-13 and the NF-κBp65 signalling pathway in KOA pathogenesis. A total of 100 KOA patients were enrolled in our hospital from December 2015 to December 2017 and were classified into either a mild KOA group (Outerbridge grade 1 and 2) or a severe KOA group (Outerbridge grade 3 and 4). Non-OA patients were included as controls. Synovial tissues from patients in both groups were collected for detection of the mRNA and protein expression of MMP-13, NF-κBp65 and IL-lß. Synovial tissue slices were subjected to haematoxylin and eosin staining and immunohistochemistry (SP method). Cartilage tissues were observed under a light microscope after Safranin O-fast green staining. Reverse transcription-quantitative PCR and western blot analyses demonstrated that the expression of MMP-13, NF-κBp65 and IL-lß in the mild and severe groups were substantially upregulated compared with the control group (all P<0.05). A positive correlation between MMP-13 and NF-κBp65 expression in the KOA synovial tissues was identified (P<0.05). Immunohistochemistry revealed that the expression of MMP-13 and NF-κBp65 was related to the severity of KOA (MMP-13: severe, 92.54%; moderate, 76.52%; control: 32.14%; and NF-κBp65: severe, 85.56%; moderate, 48.12%; control: 28.32%). This evidence indicated that the severity of KOA was related to MMP-13 and NF-κBp65 expression. The NF-κB signalling pathway may be activated during OA progression, which could upregulate the expression of MMP-13 and IL-1ß and accelerate the deterioration of articular cartilage.

[Fermentation optimization based on cell self-adaptation to environmental stress - a review].

In industrial fermentation processes, bacteria have to adapt environmental stresses. Sometimes, such a self-adaption does not work and will cause fermentation failures, although such adaptation also can generate unexpected positive effects with improved fermentation performance. Our review introduces cell self-adaption to environmental variations or stress, process optimization based on such self-adaptions, with heterologous proteins production by Pichia pastoris and butanol fermentation as examples. Our review can sever as reference for fermentation optimization based on cell self-adaption.

Evaluation of the sub-optimal induction strategies for heterologous proteins production by Pichia pastoris Mut+/MutS strains and related transcriptional and metabolic analysis.

Heterologous proteins induction by methylotrophic recombinant Pichia pastoris is generally implemented at high cells density condition. Methanol concentration (MeOH) and dissolved oxygen concentration (DO) are two crucial operating parameters controlling proteins production. It is difficult to control MeOH/DO at their desired levels simultaneously due to the extremely high oxygen consumption features. Methanol utilization plus (Mut+) and slow (MutS) strains are the two typical phenotypes of recombinant P. pastoris with quite different dynamic characteristics. Therefore, different MeOH/DO combinational control strategies or sub-optimal induction strategies could be adopted. Environments of "high MeOH/low DO" and "high DO/low MeOH" are the realistic induction strategies. In this study, we summarized our own experimental results (using Mut+/MutS strains to produce human serum albumin-human granulocyte colony stimulating factor-HSA-GCSFm/porcine interferon-α-pIFN-α), and compared to data from the literature using the above mentioned two induction strategies. The results suggested that, heterologous proteins production by Mut+ strains favors "high DO/low MeOH (DO ~ 10%, MeOH ~ 0 g/L)" induction condition, while proteins production by MutS strains prefers "high MeOH/low DO (MeOH 5-10 g/L, DO ~ 0%)" induction environment. Thus, based on the P. pastoris types, the corresponding sub-optimal induction strategies should be applied accordingly. The related metabolic analysis indicating methanol utilizing efficiency and the transcriptional analysis reflecting gene up- or down-regulations involved in several key routes in methanol and sorbitol metabolism were implemented. The analysis results strongly supported the conclusions of using the proposed sub-optimal induction strategies for different heterologous proteins production by Mut+ and MutS strains.

Correction: Enhancing monellin production by Pichia pastoris at low cell induction concentration via effectively regulating methanol metabolism patterns and energy utilization efficiency.

[This corrects the article DOI: 10.1371/journal.pone.0184602.].

Adventures in Atropisomerism: Total Synthesis of a Complex Active Pharmaceutical Ingredient with Two Chirality Axes.

A strategy to prepare compounds with multiple chirality axes, which has led to a concise total synthesis of compound 1A with complete stereocontrol, is reported.

Combinatorial synthetic pathway fine-tuning and comparative transcriptomics for metabolic engineering of Raoultella ornithinolytica BF60 to efficiently synthesize 2,5-furandicarboxylic acid.

The compound 5-hydroxymethylfurfural (HMF) has attracted much attention due to its versatility as an important bio-based platform chemical. Here, we engineered Raoultella ornithinolytica BF60 as a whole-cell biocatalyst for a highly efficient synthesis of 2,5-furandicarboxylic acid (FDCA) from HMF. Specifically, various expression cassettes of key genes, such as hmfH (gene encoding HMF/furfural oxidoreductase [HmfH]) and hmfo (gene encoding HMF oxidase), were designed and constructed for fine-tuning FDCA synthesis from HMF. The FDCA titer reached 108.9 mM with a yield of 73% when 150 mM HMF was used as the substrate. This yield was 16% higher than that without balancing key gene expression in FDCA synthetic pathways. Additionally, to strengthen HmfH expression at the translational level, ribosomal binding site (RBS) sequences, which were computationally designed using the RBS calculator, were assembled into HmfH expression cassettes. The HmfH expression in the presence of these sequences enhanced FDCA titer to 139.6 mM with a yield of 93%. Next, previously unknown candidate genes, such as aldR, dkgA, akR, AdhP1, and AdhP2, which encode enzymes that catalyze the reactions leading to the formation of the undesired product 2,5-bis(hydroxymethyl)furan (HMF alcohol) from HMF, were identified by RNA-sequencing-based transcriptomics. Combinatorial deletion of these five candidate genes led to an 88% reduction in HMF alcohol formation and 12% enhancement in FDCA production (175.6 mM). Finally, FDCA synthesis was further improved by the substrate pulse-feeding strategy, and 221.5 mM FDCA with an 88.6% yield was obtained. The combinatorial synthetic pathway fine-tuning and comparative transcriptomics approach may be useful for improving the biocatalysis efficiency of other industrially useful compounds.

[Optimizing carbon/energy metabolism to enhance monellin production by Pichia pastoris].

In heterologous protein productions by Pichia pastoris, methanol induction is generally initiated when cell density reaches very high level. However, this traditional strategy suffers with the problems of difficulty in DO control, toxic by-metabolites accumulation and low targeted protein titer. Therefore, initiating methanol induction at lower cell concentration is considered as an alternative strategy to overcome those problems. However, the methanol/energy regulation mechanisms of initiating induction at lower concentration are not clear and seldom reported. In this article, with monellin production as a prototype, we analyzed the methanol/energy metabolism in protein expression process using the strategies of initiating induction at both higher/lower cells concentrations. We attempted to interpret the advantages of the "alternative" strategy, via online measurements of methanol consumption, CO2 production and O2 uptake rates. When adopting this "alternative" strategy and maintaining temperature at 30 °C, carbon flux ratio directing into monellin precursors synthesis reached the highest level of 65%. In addition, monellin synthesis was completely associated with cell growth.

Improved production of 2,5-furandicarboxylic acid by overexpression of 5-hydroxymethylfurfural oxidase and 5-hydroxymethylfurfural/furfural oxidoreductase in Raoultella ornithinolytica BF60.

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block and can be produced by biotransformation of 5-hydroxymethylfurfural (HMF). To improve the FDCA production, two genes-one encoding HMF oxidase (HMFO; from Methylovorus sp. strain MP688) and another encoding for HMF/Furfural oxidoreductase (HmfH; from Cupriavidus basilensis HMF14)-were introduced into Raoultella ornithinolytica BF60. The FDCA production in the engineered whole-cell biocatalyst increased from 51.0 to 93.6mM, and the molar conversion ratio of HMF to FDCA increased from 51.0 to 93.6%.

Electron receptor addition enhances butanol synthesis in ABE fermentation by Clostridium acetobutylicum.

The techniques for enhancing butanol production in ABE fermentation by Clostridium acetobutylicum generally focus on adding electron carrier to strengthen NADH synthesis, repressing hydrogenase by aerating CO, supplementing butyrate, etc. However, those methods suffer from the problems of total solvent decrease, high purification cost, using expensive supplemental substances, etc. In this study, we added small amount of electron receptors (Na2SO4/CaSO4, 2g/L) into ABE fermentation broth: to alter electron/proton distributions in the intracellular electron transport shuttle system, directing more electron/proton pairs into NADH synthesis route; to stimulate intracellular accumulation of those amino acids favorable for cells survival/butanol synthesis. In ABE fermentation in a 7L fermentor, adding 2g/L Na2SO4 could raise butanol concentration to a higher level of 12.96g/L, which was 34.8% higher than that of the control. Addition of tiny amount cheap electron receptor would provide a new way to enhance bio-butanol production.

Enhanced 2,5-Furandicarboxylic Acid (FDCA) Production in Raoultella ornithinolytica BF60 by Manipulation of the Key Genes in FDCA Biosynthesis Pathway.

The compound 2,5-furandicarboxylic acid (FDCA), an important bio-based monomer for the production of various polymers, can be obtained from 5-hydroxymethylfurfural (HMF). However, efficient production of FDCA from HMF via biocatalysis has not been well studied. In this study, we report the identification of key genes that are involved in FDCA synthesis and then the engineering of Raoultella ornithinolytica BF60 for biocatalytic oxidation of HMF to FDCA using its resting cells. Specifically, previously unknown candidate genes, adhP3 and alkR, which were responsible for the reduction of HMF to the undesired product 2,5-bis(hydroxymethyl)furan (HMF alcohol), were identified by transcriptomic analysis. Combinatorial deletion of these two genes resulted in 85.7% reduction in HMF alcohol formation and 23.7% improvement in FDCA production (242.0 mM). Subsequently, an aldehyde dehydrogenase, AldH, which was responsible for the oxidation of the intermediate 5-formyl-2-furoic acid (FFA) to FDCA, was identified and characterized. Finally, FDCA production was further improved by overexpressing AldH, resulting in a 96.2% yield of 264.7 mM FDCA. Importantly, the identification of these key genes not only contributes to our understanding of the FDCA synthesis pathway in R. ornithinolytica BF60 but also allows for improved FDCA production efficiency. Moreover, this work is likely to provide a valuable reference for producing other furanic chemicals.

Enhancing monellin production by Pichia pastoris at low cell induction concentration via effectively regulating methanol metabolism patterns and energy utilization efficiency.

In heterologous protein productions by P. pastoris, methanol induction is generally initiated when cell concentration reaches very high density. The alternative strategy by initiating methanol induction at lower cells concentration was also reported to be effective in easing DO control, reducing toxic by-metabolites accumulation and increasing targeted proteins titers. However, the methanol/energy regulation mechanisms are seldom reported. We theoretically analyzed the methanol/energy metabolisms in protein expression process with the strategies of initiating induction at higher or lower cells concentrations, using monellin production as a prototype. When initiating induction at lower cells concentration and controlling induction temperature at 30°C, monellin concentration reached the highest levels of 2.62~2.71 g/L, which was 2.5~4.9 fold of those obtained with the strategy of initiating induction at higher cells concentration. With the desired induction strategy, 1) carbon metabolism ratio directing into the precursors synthesis route for monellin production reached the highest level of 65%, carbon metabolism ratios towards to precursors synthesis and ATP regeneration routes were regulated at relatively balanced levels; 2) monellin synthesis was completely cell growth associated, with the largest associated coefficient and higher specific growth rate; 3) theoretical NADH (energy) utilization efficiency η was the highest, and η stayed high levels (≥0.8) during most period (89%) within induction phase to supply sufficient energy in supporting monellin synthesis.

Microbial production of poly-γ-glutamic acid.

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on L-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid L-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.