We report the improved production of recombinant proteins in E. coli , reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales.Expression levels using this approach have reached as high as 55% of total cellular protein.Initial use of the method in instrumented fed batch fermentations enables cell densities of~30 grams dry cell weight (gCDW) per liter and protein titers up to 8.1+/-0.7 g/L (~270 mg/gCDW).The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 L (384 well plates), 100 L (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach~5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.

A key challenge in synthetic biology is the successful utilization of characterized parts, such as promoters, in different biological contexts. We report the robustness testing of a small library of E. coli PhoB regulated promoters that enable heterologous protein production in two-stage cultures. Expression levels were measured both in a rich Autoinduction Broth as well as a minimal mineral salts media. Media dependent differences were promoter dependent. 4 out of 16 promoters tested were identified to have tightly controlled expression which was also robust to media formulation. Improved promoter robustness led to more predictable scale up and consistent expression in instrumented bioreactors. This subset of PhoB activated promoters, useful for two-stage autoinduction, highlight the impact of the environment on the performance of biological parts, and the importance of robustness testing in synthetic biology.

Industrial biotechnology can lead to new routes and potentially to more sustainable production of numerous chemicals. We review the potential of biobased routes from sugars to the large volume commodity, methacrylic acid, involving fermentation based bioprocesses. We cover the key progress over the past decade on direct and indirect fermentation based routes to methacrylic acid including both academic as well as patent literature. Finally, we take a critical look at the potential of biobased routes to methacrylic acid in comparison with both incumbent as well as newer greener petrochemical based processes.

We report improved release of recombinant proteins in Escherichia coli, which relies on combined cellular autolysis and DNA/RNA autohydrolysis, conferred by the tightly controlled autoinduction of both phage lysozyme and the nonspecific DNA/RNA endonuclease from Serratia marcescens. Autoinduction occurs in a two‐stage process wherein heterologous protein expression and autolysis enzymes are induced upon entry into stationary phase by phosphate depletion. Cytoplasmic lysozyme and periplasmic endonuclease are kept from inducing lysis until membrane integrity is disrupted. After cell harvest, the addition of detergent (0.1% Triton X‐100) and a single 30 min freeze‐thaw cycle results in >90% release of protein, green fluorescent protein. This cellular lysis is accompanied by complete oligonucleotide hydrolysis. The approach has been validated for shake flask cultures, high‐throughput cultivation in microtiter plates, and larger scale stirred‐tank bioreactors. This tightly controlled system enables robust growth and resistance to lysis in routine media when cells are propagated and autolysis/hydrolysis genes are only induced upon phosphate depletion.

We report the improved production of recombinant proteins in E. coli , reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales.Expression levels using this approach have reached as high as 55% of total cellular protein.Initial use of the method in instrumented fed batch fermentations enables cell densities of 10 grams dry cell weight (gCDW) per liter and protein titers up to 2.7+/-0.2 g/L (270 mg/gCDW).The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 L (384 well plates), 100 L (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach~5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation Highlights :• Stationary phase protein expression results in high titers.• Autoinduction by phosphate depletion enables protein titers from 2-3 g/L.• Autoinduction has been validated from 384 well plates to instrumented bioreactors.