Abstract: A vector for high-level expression of a heterologous gene in Bacillus, including a DNA sequence which encodes all or part of the structural gene and contains the promoter and ribosome binding site of a Bacillus coagulans amylase gene; within or immediately downstream of the structural gene is a site for insertion of heterologous DNA; when inserted, the heterologous DNA is in the same translational reading frame as the amylase gene; the heterologous DNA thus is under control of the B. coagulans amylase gene regulatory elements and expressed at high levels. A signal-encoding sequence may be inserted independent of, or along with, the heterologous DNA leading to authentic secretion of the heterologous protein.

Abstract: A method of controlling of cell mass by starving cells that are ppc.sup.- and pc.sup.- for tricarboxylic acid cycle (TAC) intermediates, while fermenting a product compound. The cells are adapted to enable or enhance conversion of a raw material to the product via a bioconversion pathway that does not include any TAC intermediates, and preferably they are either conditionally or permanently incapable of producing a net increase in TAC intermediates via any anaplerotic pathways. First the cells are cultured to the desired cell mass under growth medium conditions characterized by the presence of at least one TAC intermediate, or by the presence of a compound that the cells can convert to a TAC intermediate via an anaplerotic pathway under the conditions present in the growth medium. In that way the medium provides the cell enough TAC intermediates to support growth.

Abstract: A vector including a DNA sequence encoding a secretory signal sequence substantially identical to the secretory signal-encoding sequence of a glucoamylase gene from Saccharomyces diastaticus or S. cerevisiae; and upstream from the signal-encoding sequence, a DNA sequence capable of promoting transcription in yeast (e.g., a high yield promoter, such as the promoter of the triose phosphate isomerase gene), transcription of the signal-encoding sequence being under the control of the transcription-promoting sequence, a site for the insertion into the vector of a heterologous DNA sequence, in reading frame with the signal-encoding sequence. The vector is useful as an expression vector in yeast.

Abstract: A bacterial cell transformed with a gene encoding a desired product and with an enhancing DNA sequence capable of enhancing the production of the desired product in the bacterial cell, the enhancing DNA sequence being further characterized in that it is capable of enhancing the production of an endogenous protease in a Gram-positive bacterial cell.

Abstract: A method of continuous product formation using at least two continuous fermentation units and a microorganism capable of being induced, in response to environmental conditions, to undergo a genetic alteration from a state favoring microorganism growth to a state favoring product production by the microorganism. The first continuous fermentation unit is maintained at environmental conditions selected to favor growth of the microorganism and to be nonpermissive for the genetic alteration. The microorganism is grown continuously in the first unit, and a portion of the growing microorganism cell mass is transferred via connecting means to the second continuous fermentation unit. Either the connecting means or the second unit is maintained at second environmental conditions selected to effect the genetic alteration. The altered microorganism is cultured in the second unit.

Abstract: An expression vector having two structural genes that form a synthetic operon expressed under the control of a single regulatory sequence. The operon genes correspond to the structural component of naturally occurring genes whose expression is controlled by distinct separate regulatory sequences. The operon genes code for enzymes in a biosynthetic pathway for producing a desired compound, and at least one of those operon genes is feedback derepressed. The vector is used to transform host cells that are cultured to produce the desired product.

Abstract: A vector including a DNA sequence encoding a secretory signal sequence substantially identical to the secretory signal encoding sequence of the Bacillus licheniformis .alpha.-amylase gene; upstream from the signal-encoding sequence, a promoter sequence and a ribosome binding site sequence, transcription of the signal-encoding sequence being under the control of the promoter sequence; and downstream from the signal-encoding sequence, a site for the insertion into the vector of a heterologous DNA sequence, in reading frame with the signal-encoding sequence.

Abstract: Proteins having chorismate mutase-prephenate dehydratase (CMPD) activity, but lacking phenylalanine sensitivity are produced by genetic engineering. The proteins contain a sequence substantially corresponding to the N-terminal 337 amino acids of Escherichia coli CMPD. Expression vectors including genes coding for those proteins and regulatory DNA enabling their expression are used to transform host microorganisms, which are cultured to produce phenylalanine.

Abstract: Cloning vectors are disclosed to obtain secretion of a desired protein from a host Streptomyces when a structural gene coding for the protein is inserted into the vector. The vector has: (1) regulatory DNA that includes a promoter sequence effective to start transcription in the host Streptomyces and DNA that encodes a ribosome-binding site; (2) a DNA sequence that codes for a signal sequence that occurs naturally in a Streptomyces strain or that derives from such a DNA sequence; and (3) at least one engineered restriction endonuclease recognition site positioned for the insertion of a structural gene, the DNA that encodes a signal sequence and attached structural gene being transcribed and translated together under the control of the regulatory DNA. Expression vectors are disclosed which include a structural gene coding for a desired protein so positioned. Streptomyces cells containing the vector and methods of using them to produce the desired protein are disclosed.

Abstract: An Escherichia coli chromosomal DNA segment is engineered for controllable excision and loss from the E. coli cell population. The DNA segment has a gene determining a function that is lost in the absence of that chromosomal segment. Specifically, the segment includes a pair of lambdoid phage att sites positioned at opposite ends thereof, and a gene encoding the function. The E. coli cell includes DNA encoding lambdoid phage int and xis, positioned for transcription under control of an external stimulus. By excising the chromosomal DNA segment once a desired cell population is achieved, cell growth and diversion of intermediates from a desired fermentation product are diminished or avoided.

Abstract: In vivo regulation of protein production is achieved by rearranging DNA segments comprising a protein-producing gene (i.e., protein-encoding DNA as well as regulatory DNA to effect the expression of the protein-encoding DNA in the host), in response to a change in an environmental condition such as temperature. The rearrangement is synchronized and directional (irreversible) in members of the cell population, because it is catalyzed by a lambda phage site-specific recombination enzyme system that operates on a pair of lambda phage attachment sites to rapidly drive the rearrangement and to avoid the reverse reaction. The cells include means to produce the lambda enzyme system in response to the change in environmental condition. By engineering one of the attachment sites within the gene that produces the protein whose production is to be regulated (yielding two gene segments), the synchronized rearrangement operates to change the gene from one configuration to another.

Abstract: A method of recovering an amino acid from a mixture by providing a source of bivalent metal ions capable of forming a complex with the amino acid, the complex being insoluble at least in a particular pH range. The complex is separated from the mixture and then dissociated, the metal ions thereby being removed from the amino acid. The amino acid may be recovered continuously from a fermentation broth of microorganisms.